We propose to apply the approaches of the organic chemist as well as methods of the enzymologist to probe the nature of the active sites of both heart and skeletal muscle creatine kinases, isoenzymes which use creatine, phosphocreatine, ADP and ATP as substrates. These creatine kinase isoenzymes thus serve a dual role as objects for screening creatine, phosphocreatine and nucleotide analogs of potentially great interest in other biological systems. They also serve as prototypal kinases, the mechanism of action for none of which is yet well understood. Our general approach involves probing the isoenzymes' active sites with synthetic substrate analogs both to see which functional groups of the substrate are most vital to activity and to see what additional groups may be added without severe detrimental effect on activity. Various physical methods, including kinetic studies and nuclear magnetic resonance studies, are proposed in this probing process. X-Ray crystallography will be used to determine the crystal structure of the enzyme. The results of these specificity studies will be used in the design of affinity labels which are analogs of the normal substrates. Affinity labels for creatine kinases are desirable, not only as probes for side-chain residues in the active-site region, but also to enable use to inhibit the isoenzymes in their natural habitat with high selectivity, so that, by difference, their physiological roles can be further elucidated. Moreover, comparisons will be made between the substrate specificities of the creatine kinase isoenzymes from both skeletal muscle and heart tissues. It should be possible to design methods of detecting one isoenzyme in the presence of others based on these specificity differences. This could have clinical value in the detection of myocardial infarctions and other muscular disorders such as muscular dystrophy.